Scintigraphic imaging and similar radiographic techniques for visualizing tissues in vivo are finding ever-increasing application in biological and medical research and in diagnostic and therapeutic procedures. Generally, scintigraphic procedures involve the preparation of radioactive agents which upon introduction to a biological subject, becomes localized in the specific organ, tissue or skeletal structure of choice. When so localized, traces, plots or scintiphotos depicting the in vivo distribution of radiographic material can be made by various radiation detectors, e.g., traversing scanners and scintillation cameras. The distribution and corresponding relative intensity of the detected radioactive material not only indicates the space occupied by the targeted tissue, but also indicates a presence of receptors, antigens, aberrations, pathological conditions, and the like.
In general, depending on the type of radionuclide and the target organ or tissue of interest, the compositions comprise a radionuclide, a carrier agent designed to target the specific organ or tissue site, various auxiliary agents which affix the radionuclide to the carrier, water or other delivery vehicles suitable for injection into, or aspiration by, the patient, such as physiological buffers, salts, and the like. The carrier agent attaches or complexes the radionuclide to the peptide carrier agent, which results in localizing the radionuclide being deposited in the location where the carrier agent concentrates in the biological subject.
Triamidethiolate and diamidedithiolate ligands have been used successfully for radiolabeling macromolecules. In general, amide-thiolate systems require harsh (75.degree. C.-100.degree. C.) radiolabeling conditions for preparing Tc and Re complexes. Under these conditions, the stability and biological properties of the small and medium bioactive peptides are often degraded.
In order to avoid harsh labeling conditions, pre-formed complexes have been coupled to the protein with some success. See Fritzberg et al., U.S. Pat. Nos. 4,965,392 and 5,037,630 incorporated herein by reference. In the "pre-formed approach," the ligand is complexed with the radionuclide and then conjugated to the bioactive peptide. A major disadvantage of the pre-formed approach is that the end user must perform both the radiolabeling step and the coupling step (attaching the complex to the bioactive peptide). The final product must be purified prior to administration. In the case of small and medium sized peptides, the metal-complex may potentially react with "active sites" of the peptide. Thus, site specific attachment of a ligand to a bioactive molecule is only possible with post formed complexes.
In the conventional "post-formed approach," the ligand is first conjugated to the peptide and the resulting conjugate is labeled with the radioisotope under complex forming conditions. In the present invention, the post-formed approach has the additional advantage of allowing preparation of the conjugated bioactive peptide in kit form. The end users would perform only the radiolabeling step.
It has been found that the presence of free thiol (instead of protected thiol) and/or replacement of an amide with an amine causes labeling of N.sub.2 S.sub.2 and N.sub.3 S ligands to proceed under milder conditions, but at the expense of some complex stability. See Rao et al., "Tc-Complexation of N.sub.2 S.sub.2 Monoaminemonoamides," Int. J. Radiat. Part B, (1991) (in press). In addition, Misra et al., "Synthesis of a Novel Diaminodithiol Ligand for Labeling Proteins and Small Molecules with Technetium-99m," Tetrahedron Letters, Vol. 30, No. 15, pp. 1885-88 (1989) and Baidoo et al., "Synthesis of a Diaminedithiol Bifunctional Chelating Agent for Incorporation of Technetium-99m into Biomolecules," Bioconjugate Chemistry, Vol. 1, pp. 132-37 (1990), report that diaminedithiol (DADT) ligands label with .sup.99m Tc at ambient temperatures.
Gustavson et al., "Synthesis of a New Class of Tc Chelating Agents: N.sub.2 S.sub.2 Monoaminemonoamide (MAMA) Ligands," Tetrahedron Letters, Vol. 32, No. 40, pp. 5485-88 (1991), compares the radiolabeling efficiency of a N.sub.2 S.sub.2 -diamidedithiol (DADS) ligand with a N.sub.2 S.sub.2 -monoamine amide (MAMA) ligand. It was found that substitution of the amide nitrogen in the DADS ligand with an amine nitrogen in the MAMA ligand produced a threefold increase in radiochemical yield when labeling with .sup.99m Tc at 37.degree. C. for 30 minutes. ##STR1##
Notwithstanding the improved metal complex formation kinetics reported with amine-containing N.sub.2 S.sub.2 and N.sub.3 S ligands, Tc and Re amide-thiolate complexes assure maximum in vivo stability and inhibit metal oxidation to the pertechnetate or perrhenate oxidation state.
From the foregoing, what is needed in the art is an amide-thiolate ligand with improved complex formation kinetics which can be labeled under mild conditions and which has excellent in vivo complex stability.